|
| Status |
Public on Jul 01, 2014 |
| Title |
RUNX1 mutant, Biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
differentiated iPS clone
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: RUNX1 mutant
|
| Treatment protocol |
No treatments were performed.
|
| Growth protocol |
Human iPSCs were derived using the Yamanaka episomal vector system1. Briefly, patient fibroblasts were reprogrammed using three episomal vectors pCXCLE-hOCT3/4-shp53, pCXCLE-hSK, and pCXLE-hUL (all generous gifts from Dr. Yamanaka). Cultured fibroblasts were nucleofected using the nucleofector II system with the NHDF solution (AMAXA, now part of Lonza). Nucleofected fibroblasts were plated on an irradiated primary mouse embryonic fibroblast feeder layer and cultured as described previously1. Once established, iPSC lines were adapted to a feeder free system, and cultured on Matrigel (BD Bioscience) in Nutristem (Stemgent), or the Essential 8 media (Life Technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (including small and long RNAs) was extracted from cultured cells after 14 days of differentiation using miRNeasy Mini kit (Qiagen). (cat. no. 217004)
|
| Label |
Biotin
|
| Label protocol |
For RNA labeling, 300 nanograms of total RNA was used in conjunction with the Affymetrix recommended protocol GeneChip WT Terminal Labeling and Controls Kit (Cat#901525) and Ambion WT Expression Kit (cat#4411973)
|
| |
|
| Hybridization protocol |
The hybridization cocktail containing the fragmented and Biotin labeled cRNAs was hybridized to The Affymetrix Human Gene 2.0 ST GeneChip. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA).
|
| Scan protocol |
Genechips were scanned using an Affymetrix Gene Chip Scanner 3000.
|
| Description |
Gene expression data from mutant RUNX1
|
| Data processing |
Gene expression intensities were calculated using GeneChip® Command Console® Software (AGCC) and Expression Console™ Software.
|
| |
|
| Submission date |
Jan 22, 2014 |
| Last update date |
Oct 21, 2014 |
| Contact name |
abdel G Elkahloun |
| E-mail(s) |
abdel@mail.nih.gov
|
| Phone |
301 402 3170
|
| Organization name |
NHGRI-NIH
|
| Lab |
MICROARRAY CORE
|
| Street address |
50, SOUTH DRIVE
|
| City |
BETHESDA |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL16686 |
| Series (1) |
| GSE54295 |
Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects |
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